Split peroxidases and methods of use

ABSTRACT

An imaging method utilizing a split peroxidase is described herein. Imaging methods involve contacting a cell with a split peroxidase and a substrate thereof to allow conversion of a substrate into a product via an enzymatic reaction catalyzed by the reconstitute split peroxidase. Also disclosed herein are split peroxidases, related products and kits.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/157,281, entitled “SPLIT PEROXIDASES AND METHODS OF USE” filed onJan. 16, 2014, which claims priority under 35 U.S.C. § 119(e) to U.S.provisional application Ser. No. 61/753,408, filed Jan. 16, 2013, thecontents of which are incorporated herein in their entirety.

GOVERNMENT SPONSORSHIP

This invention was made with Government support under Grant No. DP1GM105381 awarded by the National Institutes of Health. The Governmenthas certain right in the invention.

BACKGROUND OF INVENTION

Electron microscopy (EM) offers far better spatial resolution thanfluorescence microscopy and therefore is a very important tool for cellbiology. For example, mitochondria are just a few pixels wide byfluorescence but details and sub compartments can be seen by EM. Twoexisting genetically encoded reporters for EM are Horse RadishPeroxidase (HRP) and mini-singlet oxygen generator (SOG), bothgenerating contrast by catalyzing the polymerization of adiaminobenzidine (DAB) into an osmiophilic polymer. Photo oxidation ofminiSOG requires laser and blown oxygen. As such, use of miniSOG as anEM reporter is limited to small fields of view. HRP is a much easier touse, less temperamental, and more robust reporter than miniSOG, but itonly works in the secretory pathway, such as in the endoplasmicreticulum (ER) and the Golgi apparatus, or on cell surfaces. It isinactive in any other cellular compartment, e.g., cytosol, due todisruption of the four disulfide bonds in this enzyme. Ascorbateperoxidases (APX), including modified versions such as APEX, have alsobeen described as reporters for microscopy. Other reporters are prone toinactivation due to the strong fixation typically employed in EM.

SUMMARY OF THE INVENTION

The invention, in some aspects, relates to new imaging based reportersfor EM or fluorescence readouts. The present disclosure is based on theunexpected discoveries that a set of split peroxidases (for examplesplit versions of peroxidases such as HRP and APEX) can be used tosuccessfully convert various enzyme substrates (e.g., DAB and AmplexRed) into signal-releasing products (e.g., osmiophilic polymers andfluorescent dyes) in a number of different specific subcellularcompartments (e.g., cytosol and mitochondria) when they arereconstituted, indicating that these split enzymes are cytosolicallyactive and therefore are useful in microscopy imaging, particularly inEM imaging.

An imaging method is provided according to aspects of the invention. Themethod involves providing a sample containing a cell that expresses asplit peroxidase comprising two or more separate components of aperoxidase, and contacting the sample with a peroxidase substrate toallow conversion of the peroxidase substrate into a product via anenzymatic reaction catalyzed by a reconstituted peroxidase that formswhen the two or more components of the split peroxidase interact,wherein the product releases a detectable signal. In some embodimentsthe signal is detectable by a microscope, such as by electron microscopyor fluorescence microscopy. In other embodiments the signal isdetectable by chemiluminescence or visualization by the eye.

In some aspects the invention is an imaging method, which involvesproviding a sample containing a cell that expresses a split peroxidasecomprising two or more separate components of a peroxidase, andcontacting the sample with a peroxidase substrate to allow conversion ofthe peroxidase substrate into a product via an enzymatic reactioncatalyzed by a reconstituted peroxidase that forms when the two or morecomponents of the split peroxidase interact, wherein the productreleases a signal detectable by a microscope. In some embodiments thesignal is detected by electron microscopy. In other embodiments thesignal is detected by fluorescence microscopy.

According to other embodiments the split peroxidase is a split horseradish peroxidase (HRP). In yet other embodiments the split peroxidaseis a split ascorbate peroxidase (APX). The signal may be detected in asecretory pathway or on the cell surface. Alternatively the signal maybe detected intracellularly.

A method involving contacting a living cell with a set of splitperoxidase enzymes and a substrate under conditions suitable for thesplit peroxidase enzymes to catalyze a reaction resulting in the taggingof molecules within the vicinity of the split peroxidase enzymes isprovided according to other aspects of the invention. In someembodiments the tagged molecules comprise protein molecules. In otherembodiments the substrate is a tyramide. The tagged molecules may alsobe isolated and/or analyzed.

In certain embodiments, the method further comprises detecting thesignal under a microscope. The peroxidase substrates for use in theimaging methods described herein can be a phenol (e.g., guaiacol,pyrogallol, Amplex UltraRed, dihydrofluorescin, p-cresol, dopamine,3-methylphenol, 4-methoxyphenol, 4-hydroxybenzaldehyde, 5-aminosalicylicacid, or 4-chloro-1-naphthol) or an aniline (e.g., diaminobenzidine(DAB), 3-amino-9-ethylcarbazole, o-phenylenediamine,3,3′,5,5′-tetramethylbenzidine, o-diansidine, 5-aminosalicylic acid,Luminol, 4-aminophthalhydrazide, N-(6-Aminohexyl)-N-ethylisoluminol,N-(4-Aminobutyl)-N-ethylisoluminol, 3-methylaniline, 4-methylaniline, or4-methoxyaniline). Alternatively, the peroxidase substrate can be3-methyl-2-benzothiazolinone hydrazine or2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). When necessary,the expression of the split peroxidase or the fusion protein containingsuch can be under the control of a cell-type specific promoter.

In some examples, the imaging method described herein involvesexpression of a fusion protein that comprises a component of a splitperoxidase and a protein of interest, e.g., a mitochondrial protein,mitochondrial matrix protein, a mitochondrial intermembrane spaceprotein, a mitochondrial inner membrane protein, a mitochondrial outermembrane protein (facing cytosol), a Golgi protein, an endoplasmicreticulum lumen protein, an endoplasmic reticulum membrane protein(facing cytosol), a cell surface protein, a secreted protein, a nuclearprotein, a vesicle protein, a cell skeleton protein, a cellskeleton-binding protein, a motor protein, a gap junction protein, achromatin-organizing protein, a transcription factor protein, a DNApolymerase protein, a ribosomal protein, a synaptic protein, or anadhesion protein. In other examples, the fusion protein comprisescomponent of the split peroxidase and a cellular localization signalpeptide, such as an ER-targeting signal peptide, a Golgi-targetingsignal peptide, a mitochondria-targeting signal peptide, a nuclearlocalization signal peptide, or a nuclear export signal peptide.Examples of cellular localization signal peptides include, but are notlimited to, DPVVVLGLCLSCLLLLSLWKQSYGGG (SEQ ID NO: 4),MLATRVFSLVGKRAISTSVCVRAH (SEQ ID NO:5), LQLPPLERLTLD (SEQ ID NO:6), andKDEL (SEQ ID NO:7). When necessary, either the split peroxidase or thefusion protein can comprise a protein tag.

In some examples, the cell can be a mammalian cell, a bacterial cell, ora yeast cell. Either live cells or fixed cells can be used in theimaging method described herein for detecting a signal released from theproduct of a set of split peroxidases.

An isolated component of a split peroxidase (sometimes referred to as afragment of a peroxidase) fused to a protein of interest, optionallythrough a linker is provided according to other aspects of theinvention. The protein of interest in some embodiments is amitochondrial protein, mitochondrial matrix protein, a mitochondrialintermembrane space protein, a mitochondrial inner membrane protein, amitochondrial outer membrane protein (facing cytosol), a Golgi protein,an endoplasmic reticulum lumen protein, an endoplasmic reticulummembrane protein (facing cytosol), a cell surface protein, a secretedprotein, a nuclear protein, a vesicle protein, a cell skeleton protein,a cell skeleton-binding protein, a motor protein, a gap junctionprotein, a chromatin-organizing protein, a transcription factor protein,a DNA polymerase protein, a ribosomal protein, a synaptic protein, or anadhesion protein.

A cellular localization signal peptide linked to the split peroxidase orthe protein of interest is provided in other embodiments.

The cellular localization signal peptide may be an ER-targeting signalpeptide, a Golgi-targeting signal peptide, a mitochondria-targetingsignal peptide, a nuclear localization signal peptide, or a nuclearexport signal peptide in some embodiments. In other embodiments thecellular localization signal peptide comprises an amino acid sequenceselected from the group consisting of:

(SEQ ID NO: 4) DPVVVLGLCLSCLLLLSLWKQSYGGG, (SEQ ID NO: 5)MLATRVFSLVGKRAISTSVCVRAH,  (SEQ ID NO: 6) LQLPPLERLTLD,  and (SEQ ID NO: 7) KDEL.

In some embodiments the peroxidase has an amino acid sequence selectedfrom SEQ ID NO:1, 2, 3, 8, 9, 10, or 11, or 12 and wherein the linker isa flexible amino acid linker. The linker in some embodiments iscomprised of glycine, serine and threonine residues. In some embodimentsthe linker is a flexible 12 amino acid linker.

In other aspects, the invention is an isolated component of a splitperoxidase having a fragment of a peroxidase fused to a cellularlocalization signal. The cellular localization signal peptide in someembodiments is an ER-targeting signal peptide, a Golgi-targeting signalpeptide, a mitochondria-targeting signal peptide, a nuclear localizationsignal peptide, or a nuclear export signal peptide. The cellularlocalization signal peptide comprises an amino acid sequence selectedfrom the group consisting of:

(SEQ ID NO: 4) DPVVVLGLCLSCLLLLSLWKQSYGGG, (SEQ ID NO: 5)MLATRVFSLVGKRAISTSVCVRAH,  (SEQ ID NO: 6) LQLPPLERLTLD,  and (SEQ ID NO: 7) KDEL.In some embodiments the peroxidase has an amino acid sequence selectedfrom SEQ ID NO:1, 2, 3, 8, 9, 10, 11, or 12. In other embodiments thesplit peroxidase is SEQ ID NO: 13 or 15. The split peroxidase in otherembodiments is selected from the group consisting of amino acids 1-58 ofSEQ ID NO: 8, amino acids 1-308 of SEQ ID NO: 8, amino acids 1-213 ofSEQ ID NO: 8, amino acids 214-308 of SEQ ID NO: 8, amino acids 1-50 ofSEQ ID NO:11, amino acids 51-249 of SEQ ID NO:11, amino acids 1-200 ofSEQ ID NO:11, amino acids 201-249 of SEQ ID NO:11, amino acids 1-50 ofSEQ ID NO:12, amino acids 51-249 of SEQ ID NO:12, amino acids 1-200 ofSEQ ID NO:12, or amino acids 201-249 of SEQ ID NO:12.

According to other aspects of the invention a split peroxidase isprovided. The split peroxidase is a fragment of an APX polypeptide,wherein the APX polypeptide includes at least one amino acidsubstitution from a corresponding fragment of a naturally occurring APX.In some embodiments the naturally occurring APX has an amino acidsequence of SEQ ID NO: 10. In other embodiments, the APX includes anenhanced activity mutation (i.e. APEX, SEQ ID NO. 11). In otherembodiments the amino acid substitution is at position 133 of SEQ ID NO.11, which is optionally a proline.

In other aspects the invention is a polypeptide comprising the aminoacid sequence of SEQ ID NO: 12.

The split peroxidase used in the method described herein can be derivedfrom a peroxidase such as Horse Radish Peroxidase (HRP), an ascorbateperoxidase (APX), a yeast cytochrome c peroxidase (CCP), or a bacterialcatalase-peroxidase (BCP), which can either be a wild-type enzyme or afunctional mutant thereof.

Also within the scope of the present disclosure are any of the splitperoxidases described herein and its encoding nucleic acids (both inisolated form), as well as vectors (e.g., expression vectors in whichthe coding sequence is in operably linkage with a suitable promoter)comprising the encoding nucleic acids, and host cells (e.g., bacterialcells, yeast cells, or mammalian cells) comprising the vectors, e.g.,expression vectors for producing the peroxidase mutant. The nucleic acidencoding any of the split peroxidases as described above can be linkedin frame with a second nucleotide sequence that encodes a protein ofinterest or a cellular localization signal peptide, e.g., thosedescribed above.

An “isolated polypeptide” or “isolated polynucleotide” as used hereinrefers to a polypeptide or polynucleotide that is substantially freefrom naturally associated molecules, i.e., the naturally associatedmolecules constituting at most 20% by dry weight of a preparationcontaining the polypeptide or polynucleotide. Purity can be measured byany appropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, and HPLC.

The present disclosure also provides a method of producing any of thesplit peroxidases described herein. The method comprises culturing ahost cell that comprises an expression vector for expressing a splitperoxidase as described herein, which can be fused in frame with aprotein of interest or a cellular localization signal peptide,collecting cells thus obtained for isolation of the split peroxidase,and optionally, isolating the split peroxidase from the cultured cellsor culture medium.

A method is provided according to other aspects of the invention. Themethod involves contacting molecules in a sample or a cell with a set ofsplit peroxidase enzymes and a substrate under conditions suitable forthe split peroxidase enzymes to catalyze a reaction resulting in thetagging of the molecules within the vicinity of the split peroxidaseenzymes. In some embodiments the tagged molecules comprise proteinmolecules. In other embodiments the substrate is a tyramide. In someembodiments the split peroxidase is encoded by a nucleic acid.

The method may also include a step of isolating the tagged moleculesfrom the sample or the cell and optionally analyzing the isolatedmolecules.

In some embodiments the method is performed in a living cell. In otherembodiments the living cell is an in vitro cell or an in vivo cell in asubject.

The sample in other embodiments is a cell lysate.

The tagged molecules may be, for instance, in a synapse.

The method may be a method for detecting analytes, a method fordetecting, differentiating and/or monitoring the subcellular location ofone or more proteins in living cells, a method for detecting proteinsthat interact in defined subcellular compartments, a method for trackingthe transport of proteins through and out of the cell, a method foridentifying cell surface expression, a method for monitoring andquantifying protein secretion, and/or a method for screening formediators of localization, transport and/or secretion. In someembodiments the method is used in combination with a directed evolutionstrategy. In other embodiments the method is used for high-throughputscreening of proteins.

The proteins may be variants with modified subcellular localizationcharacteristics.

A kit is provided in other aspects of the invention. The kit includes aset of split peroxidase components and instructions for delivering thesplit peroxidase components to a cell to label one or more proteins ofthe cell. In some embodiments the kit also includes a substrate.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a diagram showing an exemplary reaction of a split HRP fordetection of a protein-protein interaction.

FIG. 2 is a set of photographs depicting staining for several differentpairs of split HRP.

FIG. 3 is a set of photographs showing split HRP staining with DAB whichgives contrast for electron microscopy.

FIGS. 4A and 4B are diagrams showing the three dimensional structure ofHRP, depicting a cut site for producing split HR. The bottom panel ofFIG. 4B is a schematic diagram depicting HRP reconstitution at synapses.

FIG. 5 is a diagram detailing the labeling of a synaptic cleft usingsplit HRP.

FIG. 6 shows a diagram depicting the schematics of split APEX constructsused for detection of protein-proteins interactions.

FIG. 7 shows the results of an enzymatic reaction where resorufin, afluorescent product, is produced from Amplex Red through the action of areconstituted split APEX in the presence of H₂O₂.

FIG. 8 shows that both split HRP fragments are required for activity.The split HRP fragments in this experiment were localized to the ERlumen using the approach described in FIG. 1, and in this case, thefragments were not attached to FRB and FKBP—the fragments were simplyfree-floating in the ER lumen in this case.

FIG. 9 demonstrates that split HRP is more sensitive than split GFP. Inthis experiment, cultured neurons were transfected and labeled asdescribed for FIG. 5.

FIG. 10 shows that inter-cellular reconstitution of HRP is dependent onthe protein-protein interaction between neuroligin (NLG) and neurexin(NRX3B).

FIG. 11 demonstrates that split HRP activity survives chemical fixationand a variety of permeabilization and tissue blocking treatments.Cultured neurons were transfected in “Cis” with the split HRP fragmentsalong with a green fluorescent protein marker.

FIG. 12 shows split APEX staining with DAB, which gives contrast forelectron microscopy. This figure is similar in concept to FIG. 3, exceptthat split APEX is used here instead of split HRP.

FIG. 13 demonstrates how split “APEX2”, which is derived from animproved version of APEX, performs much better than the original splitAPEX.

FIG. 14 shows split APEX2 using biotin-phenol as the labeling substratehas potential for proteomic labeling applications. Cells here weretransfected and treated with or without rapamycin as in FIG. 13.

FIG. 15 characterizes the kinetics of rapamycin response for splitAPEX2. HEK293T cells were transiently transfected with complementarysplit APEX2 fragments (corresponding to the cut site after amino acid89).

FIGS. 16A-16C overview how different “cut sites” are evaluated for splitperoxidases.

FIG. 17 demonstrates how different split HRP fragment pairs might bedesirable for different applications. It specifically looks attemperature and rapamycin dependence of activity for 7 fragment pairs.

DETAILED DESCRIPTION

It was discovered that, unexpectedly, split peroxidase enzymes areenzymatically active in mammalian cells and remain active after thecells have been subjected to membrane-preserving fixation, resulting inthe generation of minimally-diffusive reaction products that cannotcross membranes. Thus, split peroxidases can be used as reporters in awide variety of imaging methods for, e.g., determining protein topologywithin membranes and including in vitro and in vivo assays as well asassays in lysates and other samples.

Accordingly, described herein are imaging methods involving expressionof a split peroxidase in cells and incubation of the cells with asuitable substrate under conditions allowing conversion of the substrateinto a product that releases a detectable signal. The methods providedherein can be used to detect and differentiate the subcellular locationof a protein of interest in living cells, detect proteins that interactin defined subcellular compartments, track the transport of proteinsthrough and out of the cell, identify cell surface expression, monitorand quantify protein secretion, and screen for mediators oflocalization, transport and/or secretion. These assays may also be usedin combination with directed evolution strategies, and scaled tohigh-throughput screening of protein variants with modified subcellularlocalization characteristics. The assays are useful to visualize proteinlocalization in, for example, the synaptic regions, nucleus, cytoplasm,plasma membrane, endoplasmic reticulum, golgi apparatus, filaments ormicrotubules such as actin and tubulin filaments, endosomes, peroxisomesand mitochondria.

The split fluorescent protein systems described herein generallycomprise two or more self-complementing fragments of a peroxidase (splitperoxidases). These fragments are referred to herein as splitperoxidases or a component of a split peroxidase. Either or both or allof the fragments may be functionalized with a subcellular targetingsequence enabling it to be expressed in or directed to a particularsubcellular compartment (i.e., the endoplasmic reticulum, a synapticregion, the cytoplasm, a nucleus) and/or to a protein of interest.

For example, a polynucleotide construct encoding a fusion of a testprotein and a split peroxidase may be expressed in cells containing acomplementary split peroxidase that has been localized to thesubcellular compartment of interest. The complementary split peroxidasemay be functionalized to contain a localization signal sequence capableof directing the split peroxidase into the desired subcellularcompartment. The expressed protein-split peroxidase fusion protein willonly be able to complement with the complementary split peroxidase if itis able to gain access to the same subcellular compartment thecomplementary split peroxidase has been directed to. Thus, for example,if an ER localization signal is used, the fusion protein would belocalized to the ER. A split peroxidase localized to the ER will beavailable to self-complement and generate a signal in the ER. Thesemethods are applicable to any of the assays described herein. Forinstance, they may be used to identify proteins that localize to aparticular subcellular compartment or structure as well as to identifynovel localization signals.

In some instances, it may be desirable to have expression of the testprotein-split peroxidase either precede or lag the expression ortransfection of the complementary split peroxidase, in order toeliminate non-specific fluorescence resulting from transientlocalization of either fragment of split peroxidase in the course ofprocessing or transport to the compartment of interest. In otherinstances, it may be desirable to visualize protein transport throughthe cell over a time course, and in such instances, the two or moresplit peroxidases may be co-expressed, from one or more constructs, andoptionally under the control of individually inducible promoter systems.

The invention involves a set of complementary split peroxidases. A splitperoxidase as used herein refers to a portion of a peroxidase that isless than a whole peroxidase. A set of complementary split peroxidasesis two or more split peroxidases that together make a whole peroxidase.

Peroxidase, as used herein, refers to naturally occurring or syntheticperoxidases that use hydrogen peroxide as the electron acceptor tocatalyze a number of oxidative reactions. A naturally occurringperoxidase is a peroxidase having an amino acid sequence that is thesame as an amino acid sequence of a peroxidase found in nature. Asynthetic peroxidase is a peroxidase that has an amino acid sequencethat is the distinct from an amino acid sequence of a peroxidase foundin nature. For instance, it may include one or more substituted aminoacids. A synthetic peroxidase maintains peroxidase function. In nature,peroxidases are found in plants, fungi, and bacteria, and includemultiple subfamilies: Horse Radish Peroxidase (HRP), yeast cytochrome cperoxidase (CCP), ascorbate peroxidase (APX), and bacterialcatalase-peroxidase (BCP). CCP is a soluble protein found in themitochondrial electron transport chain in yeast, where it protects yeastcells against toxic peroxides. APX is the main enzyme responsible forhydrogen peroxide removal in chloroplasts and cytosol of higher plants.Dalton, 1991, Ascorbate peroxidase, 2:139-153. Naturally, this enzyme,around 28 kD in molecular weight, is expressed in plant cytosol. Itcontains no disulfides or Ca⁺² ions and forms dimers. BCP is a bacterialenzyme that exhibits both peroxidase and catalase activities. It isthought that catalase-peroxidase provides protection to cells underoxidative stress. Welinder, 1991, Biochim Biophys. Acta 1080(3):215-220.

Examples of wild-type peroxidases are provided in Table 1 below:

TABLE 1 Exemplary Peroxidases Genbank accession # or PDB (protein EnzymeSpecies data bank) code Ascorbate peroxidase Pisum sativum (pea)CAA43992.1 Ascorbate peroxidase Glycine max (soybean) AAA61779.1Cytochome c peroxidase Saccharomyces PDB: 2CYP cerevisiae (yeast)Leishmania major Leishmania major (a PDB: 3RIV peroxidase parasiticprotozoa) Mycobacterium Mycobacterium PDB: 1SJ2 tuberculosis catalase-tuberculosis peroxidase Horse Radish Peroxidase

Also provided below are amino acid sequences of representativeperoxidase:

Pea APX (SEQ ID NO: 1):   1 mgksyptvsp dyqkaiekak rklrgfiaek kcaplilrlawhsagtfdsk tktggpfgti  61 khqaelahga nngldiavrl lepikeqfpi vsyadfyqlagvvaveitgg pevpfhpgre 121 dkpepppegr lpdatkgsdh lrdvfgkamg lsdqdivalsgghtigaahk ersgfegpwt 181 snplifdnsy ftelltgekd gllqlpsdka lltdsvfrplvekyaadedv ffadyaeahl 241 klselgfaea S. cerevisiae CCP (SEQ ID NO: 2):  1 ttplvhvasv ekgrsyedfq kvynaialkl reddeydnyi gygpvlvrla whisgtwdkh  61 dntggsyggt yrfkkefndp snaglqngfk flepihkefp wissgdlfsl ggvtavqemq121  gpkipwrcgr vdtpedttpd ngrlpdadkd agyvrtffqr lnmndrevva lmgahalgkt181 hlknsgyegp wgaannvftn efylnllned wklekndann eqwdsksgym mlptdysliq241 dpkylsivke yandqdkffk dfskafekll engitfpkda pspfifktle egglM. tuberculosis BCP (SEQ ID NO: 3):   1mpeqhppite tttgaasngc pvvghmkypv egggnqdwwp nrinlkvlhq npavadpmga  61 afdyaaevat idvdaltrdi eevmttsqpw wpadcghygp lfirmawhaa gtyrihdgrg 121gagggmqrfa pinswpdnas ldkarrllwp vkkkygkkls wadlivfagn calesmgfkt 181fgfgfgrvdq wepdevywgk eatwlgdery sgkrdlenpl aavqmgliyv npegpngnpd 241 pmaaavdire tfrrmamndv etaalivggh tfgkthgagp adlvgpepea apleqmglgw 301 kssygtgtgk daitsgievv wtntptkwdn sfleilygye weltkspaga wqytakdgag 361agtipdpfgg pgrsptmlat dlslrvdpiy eritrrwleh peeladefak awyklihrdm 421gpvarylgpl vpkqtllwqd pvpayshdlv geaeiaslks qirasgltvs qlvstawaaa 481ssfrgsdkrg ganggrirlq pqvgwevndp dgdlrkvirt leeiqesfns aapgnikvsf 541adlvvlggca aiekaakaag hnitvpftpg rtdasgegtd vesfavlepk adgfrnylgk 601gnplpaeyml ldkanlltls apemtvlvgg lrvlganykr  lplgvfteas esltndffvn 661lldmgitwep spaddgtyqg kdgsgkvkwt gsrvdlvfgs nselralvev ygaddaqpkf 721vqdfvaawdk vmnldrfdvr wild-type Horseradish peroxidase  (SEQ ID NO: 8)QLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQ GQIRLNCRVVNSNSwild-type cytochrome c peroxidase sequence (SEQ ID NO: 9)TTPLVHVASVEKGRSYEDFQKVYNAIALKLREDDEYDNYIGYGPVLVRLAWHTSGTWDKHDNTGGSYGGTYRFKKEFNDPSNAGLQNGFKFLEPIHKEFPWISSGDLFSLGGVTAVQEMQGPKIPWRCGRVDTPEDTTPDNGRLPDADKDADYVRTFFQRLNMNDREVVALMGAHALGKTHLKNSGYEGPWGAANNVFTNEFYLNLLNEDWKLEKNDANNEQWDSKSGYMMLPTDYSLIQDPKYLSIVKEYANDQDKFFKDFSKAFEKLLENGITFPKDAPSPFIFKTLEEQGL wild-type soybean APX (SEQ ID NO: 10) GKSYPTVSADYQKAVEKAKKKLRGFIAEKRCAPLMLRLAWHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPEVPFHPGREDKPEPPPEGRLPDATKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELG FADAsoybean APX K14D, W41F, E112K (monomeric soybeanAPX with an enhanced-activity mutation)  (SEQ ID NO: 11)GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDATKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELG FADA

Examples of other APX enzymes include, but are not limited to Medicagotruncatula Cytosolic ascorbate peroxidase (e.g., GenBank accession no.XP_003606510), Vigna unguiculata cytosolic ascorbate peroxidase (e.g.,GenBank accession no. AAB038441, Glycine max L-ascorbate peroxidase 2(e.g., GenBank accession no. NP_001235587), Ziziphus jujuba ascorbateperoxidase (e.g., GenBank accession no. BAM28755), Camellia sinensisascorbate peroxidase (GenBank accession no. ABD97259), and Solanumlycopersicum cytosolic ascorbate peroxidase (e.g., GenBank accession no.NP_001234788).

Examples of other CCP enzymes include, but are not limited to,Saccharomyces cerevisiae Ccp1p (e.g., GenBank accession no. EIW09306),Saccharomyces arboricola ccp1p (e.g., GenBank accession no. EJS42830),and Saccharomyces kudriavzevii CCP1 (e.g., GenBank accession no.EJT43981). Examples of other BCP enzymes include, but are not limitedto, Mycobacterium tuberculosis catalase-peroxidase (e.g., Genbankaccession no. AAK06516 and AAA18230), Streptomyces griseoaurantiacuscatalase/peroxidase (e.g., GenBank accession no. ZP_08290983), andRhodococcus opacus catalase-peroxidase (e.g., GenBank accession no.YP_002782511).

Peroxidases in each subfamily are highly homologous across species.Thus, each subfamily of peroxidases from other yeast, plant or bacterialspecies are well known in the art and can be retrieved from, e.g.,GenBank or Protein Data Bank, using any of the above described enzymesas a query.

In addition to wild-type enzymes or naturally occurring peroxidases suchas those described above, the peroxidases described herein also includesynthetic peroxidases that are functional mutants of native enzymes. Afunctional mutant may share at least 80% sequence identity (e.g., 85%,90%, 95%, 97%, 98%, or 99%) with its wild-type counterpart and preservesthe desired enzymatic activity. The “percent identity” of two amino acidsequences is determined using the algorithm of Karlin and Altschul Proc.Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin andAltschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithmis incorporated into the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the protein molecules of theinvention. Where gaps exist between two sequences, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used.

Alternative or in addition, the enzyme mutants described herein maycontain mutations (e.g., amino acid residue substitution) at up to 20positions (e.g., up to 15, 10, or 5 positions) as relative to awild-type counterpart.

It was known in the art that mutations introduced into non-functionaldomains of an enzyme are unlikely to affect the activity of that enzyme.Accordingly, the functional mutants of peroxidases may contain mutationsin non-functional domains of a wild-type enzyme. Crystal structures of anumber of representative peroxidases have been determined already.Bertrand et al., 2004, J. Biol. Chem. 279:38991-38999; Finzel et al., J.Biol. Chem. 1984, 259:13027-13036; and Jasion et al., 2011, J. Biol.Chem. 286:24608-24615. In addition, it was known in the art that thisfamily of peroxidases is homologous across species. Thus, functionaldomains of this enzyme can be determined based on the known crystalstructures and by comparing amino acid sequences across species. Oneexample is provided below:

The structure-function correlation of pea APX (SEQ ID NO:1, GenBankaccession no. CAA43992), a representative APX, was well known in theart. For example, positions 34, 35, 38, 132-134, 145, 159, 160, 162,163, 165-169, 172, 173, 179, 205, 207, 235 and 239 are suggested asresidues involved in heme binding; positions 111, 163, 165, 166, 168,193, 202, and 203 are suggested as residues involved in substratebinding; and positions 164, 180, 182, 185, 187, and 189 are suggested asresidues involved in ion binding. Given the cross-species sequencehomology, the structure-function correlation of other APX enzymes can bereadily determined based on such correlation of pea APX.

Alternatively, conservative amino acid substitutions may be introducedinto a wild-type peroxidase to provide functionally equivalent mutants.As used herein, a “conservative amino acid substitution” refers to anamino acid substitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids within the following groups: (a) M, I, L, V;(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the peroxidase mutants described herein aremonomeric mutants of APX or BCP. A monomeric mutant as described hereinrefers to a mutant of a wild-type dimeric peroxidase (e.g., anaturally-occurring APX or CCP) that can exist in monomer form.Preferably, at least 50% (e.g., 60%, 70%, 80%, 90%, or 95%) of such amutant is present in monomer form when expressed in host cells. Suchmutants can be prepared by introducing mutations at amino acid residuesthat are involved in dimerization, which can be identified via sequencealignment with a native monomeric APX (e.g., a maize APX; see Koshiba etal., Plant and Cell Physiology 34: 713-721, 1993). In some examples,such a monomeric mutant share at least 80% (e.g., 85%, 90%, 95%, or 98%)sequence homology to a wild-type reference peroxidase (e.g., an APX orCCP).

At least residues K14, K18, R21, R24, A28, E106, E112, I185, E228, andD229 in an exemplary APX (SEQ ID NO:1) may be involved in formation ofthe dimer interface of this enzyme. It is suggested that E17 and K20 mayalso be involved in dimer formation. Thus, a monomeric mutant of thisAPX can contain mutations (e.g., amino acid residue substitutions) atone or more of these positions. For example, the following amino acidresidue substitution(s) can be introduced into SEQ ID NO:1 to produce amonomeric mutant: K14D, E17N, K20A, R21L, A28K, E112K, E228K, D229K, ora combination thereof (e.g., A28K/E112K, K14D/D229K, K14D/E228K,K14D/E112K, E112K/D229K, A28K/E112K/D229K, K14D/E112K/D229K,K14D/E112K/E228K, or A28K/E112K/E228K). Examples of monomeric mutants ofSEQ ID NO:1 include, but are not limited to, single mutant K14D, A28K,E112K, E228K, or D229K, double mutant A28K/E112K, K14D/E112K (mAPX),K14D/E228K, K14D/D229K, E112K/E228K, or E112K/D229K, triple mutantE17N/K20A/R21L, A28K/E112K/D229K, K14D/W41F/E112K, K14D/E112K/D229K,K14D/E112K/E228K, or A28K/E112K/E228K.

As used herein, “single mutant,” “double mutant,” “triple mutant,”“quadruple mutant,” “quintuple mutant,” etc. refer to mutants containingonly the 1, 2, 3, 4, 5, etc. defined amino acid residue substitutions ascompared to the corresponding wild-type counterpart. For example, doublemutant K14D/E112K (also designated “mAPX” in the present disclosure) isa mutant that is otherwise identical to SEQ ID NO:1 except for the K14Dand E112K substitutions and triple mutant K14D/W41F/E112K (APEX) isotherwise identical to SEQ ID NO:1 except for the three defined aminoacid residue substitutions.

Monomeric mutants of other APX enzymes can contain one or more mutations(e.g., amino acid residue substitutions) at one or more positionsinvolved in dimerization of the counterpart wild-type enzyme, e.g.,corresponding to those in SEQ ID NO:1 as described above.

The same mutagenesis strategy as described above can be applied to BCPsto generate BCP monomeric mutants.

In other embodiments, the peroxidase mutants described herein are highactivity mutants, i.e., exhibiting higher enzymatic activity(particularly towards a desirable substrate, such as DAB) as compared totheir wild-type counterpart (e.g., having an enzymatic activity at least20%, 50%, 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold,100-fold, 1000-fold, or higher than the wild-type counterpart). Such amutant can contain mutations (e.g., amino acid residue substitutions) atone or more positions involved in enzymatic activity (e.g., heme bindingsites or substrate binding sites). In some examples, such a highactivity mutant share at least 80% (e.g., 85%, 90%, 95%, or 98%)sequence homology to a wild-type reference peroxidase (e.g., an APX, aCCP, or a BCP).

In some examples, a high activity mutant of APX can be prepared bytransplanting features of the active site of HRP (which acts on DAB),e.g., the cage of hydrophobic (e.g., aromatic) side chains, into awild-type APX. W41, G69, D133, T135, and K136 in SEQ ID NO:1 mayconstitute the active site of a pea APX (SEQ ID NO:1). A high activitymutant of this APX can be prepared by replacing one or more of theseresidues with a hydrophobic (e.g., an aromatic residue such as F, Y, orW). For example, at least one of W41, G69, T135, and K136 can bereplaced with F, Y, or W. Alternatively or in addition, D133 can bereplaced with A, G, I, L, or V. Examples of high activity mutants of SEQID NO:1 can contain the following amino acid residue substitutions:W41F, G69F, W41F/G69F, D133A/T135F/K136F, W41F/D133A/T135F/K136F,G69F/D133A/T135F/K136F, and W41F/G69F/D133A/T135F/K136F.

High activity mutants of other APX enzymes can contain one or moremutations (e.g., amino acid residue substitutions) at one or morepositions involved in enzymatic activity of the counterpart wild-typeenzyme, e.g., corresponding to those in SEQ ID NO:1 as described above.In some examples, a hydrophobic residue (e.g., an aromatic residue) isintroduced into one or more of the residues important to the enzymaticactivity of the peroxidase, which can be identified by comparing theamino acid sequence of the wild-type enzyme with SEQ ID NO:1.

In other embodiments, high activity mutants of a CCP enzyme can beprepared by introducing mutations (e.g., amino acid residuesubstitutions) at one or more active sites of a reference yeast CCP,e.g., positions corresponding to W51, S81, D146, D148, K149, and G186 inSEQ ID NO:2. In some examples, one or more positions corresponding toW51, S81, D148, K149, and G186 in SEQ ID NO:2 are replaced with ahydrophobic residue (e.g., an aromatic residue such as F, Y, or W) toproduce a high activity mutant. Alternatively or in addition, theresidue at the position corresponding to D146 in SEQ ID NO:2 can bereplaced with a hydrophobic residue such as A, G, V, I, and L.

In yet other embodiments, high activity mutants of a BCP enzyme can beprepared by introducing mutations (e.g., amino acid residuesubstitutions) at one or more active sites of a reference wild-type BCP,e.g., positions corresponding to W107, D137, E223, N231, and G316 in SEQID NO:3. In some examples, one or more positions corresponding to W107,D137, E223, and G316 in SEQ ID NO:3 are replaced with a hydrophobicresidue (e.g., an aromatic residue such as F, Y, or W) to produce a highactivity mutant. Alternatively or in addition, the residue at theposition corresponding to N231 in SEQ ID NO:3 can be replaced with ahydrophobic residue such as A, G, V, I, and L.

Further, the peroxidase mutants described herein can contain both one ormore mutations leading to monomer formation and one or more mutationsleading to elevated enzymatic activity. Such a mutant can contain anycombination of the monomeric mutations and high activity mutationsdescribed herein. For example, such an APX mutant can contain acombination of (a) K14D, E112K, E228K, D229K, K14D/E112K, K14D/E228K,K14D/D229K, E17N/K20A/R21L, or K14D/W41F/E112K, and (b) G69F, G174F,W41F/G69F, D133A/T135F/K136F, W41F/D133A/T135F/K136F,G69F/D133A/T135F/K136F, or W41F/G69F/D133A/T135F/K136F. In someexamples, the just-described APX mutant can be a combination of (a)single mutant K14D, single mutant E112K, single mutant E228K, singlemutant D229K, double mutant K14D/E112K, double mutant K14D/E228K, doublemutant K14D/D229K, triple mutant E17N/K20A/R21L, or triple mutantK14D/W41F/E112K, and (b) single mutant W41F, single mutant G69F, singlemutant G174F, double mutant W41F/G69F, triple mutant D133A/T135F/K136F,quadruple mutant W41F/D133A/T135F/K136F, quadruple mutantG69F/D133A/T135F/K136F, or quintuple mutant W41F/G69F/D133A/T135F/K136F.Examples of such combined mutants include, but are not limited to,K14D/E112K/W41F (APEX), and K14D/E112K/W41F/D133A/T135F/K136F.

An exemplary synthetic peroxidase useful according to the invention isreferred to herein as APEX (SEQ ID NO. 11). Another useful syntheticperoxidase useful according to the invention is APEX2. This enzyme hasonly 1 mutation relative to APEX, but greatly improves the brightness oflabeling for all applications tested so far. APEX2 has the followingamino acid sequence:

(SEQ ID NO: 12) GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELG FADA.

The peroxidases used according to the methods of the invention are splitperoxidases. A split peroxidase, as discussed above, is a fragment of aperoxidase, such as those described herein, including naturallyoccurring and synthetic mutant peroxidases, which together with one ormore other split peroxidases reconstitutes to form a functionalperoxidase. A split peroxidase on its own (without reconstitution) isnot enzymatically active against the substrate being used in theparticular assay. A set of peroxidases includes two or more splitperoxidases, which are separate components of a full peroxidase. In someinstances the set of peroxidases is two split peroxidases, whichtogether form the complete peroxidase. In other instances the set ofperoxidases is three, four, or five split peroxidases, which togetherform the complete peroxidase. In some embodiments the set of splitperoxidases may form less than a complete peroxidase, as long as thereconstituted peroxidase is functional.

Any of the split peroxidases as described herein can be prepared byroutine recombinant technology. In particular, the peptides can beprepared according to methods for altering polypeptide sequence known toone of ordinary skill in the art such as are found in references whichcompile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Second Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, or Current Protocols in MolecularBiology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.

Nucleic acids encoding a split peroxidase can be inserted via routinecloning technology into a vector, such as an expression vector in whichthe coding sequence is in operable linkage with a suitable promoter. Asused herein, a “vector” may be a nucleic acid into which one or moredesired sequences may be inserted by, e.g., restriction and ligation,for transport between different genetic environments or for expressionin a host cell. Vectors are typically composed of DNA although RNAvectors are also available. Vectors include, but are not limited to,plasmids, phagemids and virus genomes. A cloning vector is one which isable to replicate in a host cell, and which is further characterized byone or more endonuclease restriction sites at which the vector may becut in a determinable fashion and into which a desired DNA sequence maybe ligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase.

An expression vector can be introduced into a suitable host cell and thetransformed host cell thus obtained can be cultured under suitableconditions allowing expression of the split peroxidase. The expressedenzyme can then be isolated from the cell culture; its enzymaticactivity and monomeric property can be confirmed via methods known inthe art, e.g., SDS-PAGE, gel filtration, and an in vitro enzymaticassay.

Any of the split peroxidase as described herein, can be used in knownimaging methods including those described herein for determining variousaspects of proteins, e.g., protein topology. In general, this imagingmethod can be performed by providing a sample containing cells thatexpress a split peroxidase, a fusion protein comprising a splitperoxidase, and a protein of interest, a cellular localization signalpeptide, and/or a protein tag. This method is particularly useful instudying in live cells the structure/function of the protein ofinterest, which can be any protein, such as mammalian proteins.

It is also useful for examining protein interactions in lysates andother protein containing samples. For example, the present inventionprovides methods for rapid and sensitive assays for detectingprotein-protein, protein-nucleic acid, protein-small molecule or otherprotein-ligand interactions, and antagonists and/or agonists of such aninteraction using split monomeric protein reporter systems including,but not limited to those generating enzymatic activity, bioluminescence,chemiluminescence, fluorescence or absorbance, for example usingluciferase, β-lactamase or a fluorescent protein reporter system in acell-free assay system. The two portions of the split peroxidases cometogether in a cell-free assay and their association is mediated by aninteraction of an attached protein and its specific binding ligand,which can be an antibody or other protein, a specific nucleic acidsequence or a methylated or nonmethylated nucleic acid molecule, asingle- or double-stranded RNA molecule, a small molecule, hormone orgrowth factor, among others. Protein-ligand and protein-small moleculeinteractions can be assessed when at least one portion of the splitperoxidase is covalently or noncovalently linked to either a ligand orto an antagonist or agonist of a bimolecular interaction and the second,complementing portion of the split peroxidase is expressed in acell-free translation system. Interaction of the two binding partners,with either their ligands or each other, brings the two portions of thesplit reporter protein into sufficiently close proximity that the twoportions reassemble into a functional protein with, for example,detectable enzymatic or other activity. Antagonists or agonists of suchinteractions can be assessed by detecting the displacement of onebinding partner, and the resulting decrease in reporter signal or bydetecting enhanced interaction via increased reporter signal,respectively. Within the present methods, at least one portion of thesplit peroxidase is synthesized in an in vitro translation assay, and itmay be synthesized after in vitro transcription of the mRNA encodingthat protein.

This method is particularly useful in imaging cellular organelle (e.g.,mitochondria) in live cells.

The expression of the detection system described herein may beconstitutive or inducible. The split peroxidase may be pre-localized tothe compartment of interest, for example by inducing the expression of apolynucleotide encoding the split peroxidase, terminating induction, andthen expressing the complementary split peroxidase through a separatelyinducible system. Complementation between the pre-localized assay splitperoxidases and the expressed test protein-tag fusion results influorescence in the specific cell compartment in response to labelingwith a peroxidase substrate.

Additionally, cells may be engineered to contain a plurality ofcomplementary split peroxidases, each of which is localized to adifferent subcellular compartment. The peroxidase substrate may bedesigned or selected to produce different color fluorescence when thesplit peroxidase is reconstituted. Such an assay may be used to screenproteins for their subcellular localization profiles at fixed timepoints or in real time and to visualize protein trafficking dynamically.

Proteins may also be purified by including a modification to one of thesplit peroxidases that can be used as an affinity tag. A sequence ofamino acid residues that functionalize the split peroxidase to bind to asubstrate that can be isolated using standard purification technologiescan be used. For example, a split peroxidase may be functionalized tobind to glass beads, using chemistries well known and commerciallyavailable (e.g., Molecular Probes Inc.). Alternatively, the splitperoxidase is modified to incorporate histidine residues (HIS tags) inorder to functionalize the split peroxidase to bind to metal affinityresin beads. A HIS-tag split peroxidase can be used to purify secretedproteins from growth media using standard cobalt bead columns, andenables the quantification of soluble and insoluble protein as well asthe purification and elution of protein to 95% purity without the needfor any another purification tag system.

Multicolor labeling strategies may also be combined withfluorescence-activated cell sorting (FACS) in order to convenientlyselect and isolate cells displaying a particular fluorescence. Thispermits FACS differential sorting of different tagged mutants localizedto multiple compartments.

The methods of the invention may also be used to screen for agents thatmodulate protein localization. In one embodiment, a split peroxidasefusion is transfected into a cell, and an agent (drug) of interest isadded to the cell. Complementary split peroxidases are functionalized tobe directed to different subcellular compartments and result indifferent fluorescent colors upon complementation and exposure to aperoxidase substrate. The color of the fluorescence is determined bywhich substrate is used. The split peroxidases are expressed in ortransfected into the cell following the addition of the drug. Confocalmicroscopy is then used to examine the localization of the test protein.Indeed after complementation and substrate exposure, the changes influorescence emission after addition of the drug may also be visualized,so that changes in protein localization, due to the drug, may beobserved. The absence of fluorescence provides an indication of a directeffect on the protein's transport. Similarly, the modulating influenceof any environmental stimulus, exogenous protein, or gene may be studiedusing this assay.

The methods of the invention may also enable the detection of a proteinthat interacts with another protein in a particular subcellularcompartment. Thus, for example a protein of interest is expressed infusion with a split peroxidase such that it becomes localized to thesubcellular compartment of interest, e.g. the synapse. The localizationmay be a result of the protein's native localization signals or theresult of a localization functionality engineered into the fusionprotein. The complementary split peroxidase, functionalized to transportto the subcellular compartment of interest, is expressed in the cell ortransfected into the cell. Fluorescence detected in the cellularcompartment of interest indicates that the split peroxidasesco-localized and self-complemented in the presence of substrate, thusindicating that the test protein localizes to the compartment ofinterest and binds to the protein of interest in that compartment.

To perform the imaging methods described herein, a split peroxidase,either alone or in fusion with a protein of interest or a cellularlocalization signal peptide, is introduced into a host cell of interestfor expression via routine recombinant technology. A protein of interestcan be any protein, the topology of which is of interest. In someexamples, a protein of interest can be a subcellularcompartment-specific protein, such as a cytosol protein, mitochondrialprotein, mitochondrial matrix protein, a mitochondrial intermembranespace protein, a mitochondrial inner membrane protein, a mitochondrialouter membrane protein (facing cytosol), a Golgi protein, an endoplasmicreticulum lumen protein, an endoplasmic reticulum membrane protein(facing cytosol), a cell surface protein, a secreted protein, a nuclearprotein, a vesicle protein, a cell skeleton protein, a cellskeleton-binding protein, a motor protein, a gap junction protein, achromatin-organizing protein, a transcription factor protein, a DNApolymerase protein, a ribosomal protein, a synaptic protein, or anadhesion protein.

Cellular localization signal peptides comprises amino acid sequence thatrecognize, target, or direct the polypeptide containing such to aparticular sub-cellular component, e.g., the nucleus, cytoplasm,mitochondria, or Golgi apparatus. See: C. Dingwall et al. (1991) TIBS16:478-481. Such signal peptides are well known in the art. See, e.g.,Snapp et al., 2003, J. Cell Biol., 163(2):257-269; Perocchi et al.,2010, Nature, 467:291-297; and Uttamapinant et al., 2010, PNAS107(24):10914-10919. Various subcellular localization signal sequencesor tags are known and/or commercially available. These tags are used todirect split peroxidases to particular cellular components or outside ofthe cell. Mammalian localization sequences capable of targeting proteinsto the synapse, nucleus, cytoplasm, plasma membrane, endoplasmicreticulum, golgi apparatus, actin and tubulin filaments, endosomes,peroxisomes and mitochondria are known. Cellular localization signalpeptides for use in the present disclosure include, but are not limitedto, nuclear export signals (NES), nuclear localization signals (NLS),matrix signals, ER localization/targeting signals,mitochondrial-targeting signals, and Golgi-targeting signals. Examplesare, but are not limited to, DPVVVLGLCLSCLLLLSLWKQSYGGG (SEQ ID NO:4)(ER), MLATRVFSLVGKRAISTSVCVRAH (SEQ ID NO: 5)(mitochondria),LQLPPLERLTLD (SEQ ID NO:6)(nuclear export signal, cytosyl), KDEL (SEQ IDNO:7)(ER/Golgi), S K K E E K G R S K K E E K G R S K K E E K G R I H R I[SEQ ID NO:15], S S G E L R T G G A K D P P V A T [SEQ ID NO:16], M S VL T P L L L R G L T G S A R R L P V P R A K I H S L G D P P V A T [SEQID NO:17], M L L S V P L L L G L L G L A V A V [SEQ ID NO:18] andfunctional variants thereof, e.g., containing mutations such asconservative amino acid residue substitutions at one or more positions(e.g., up to 2, 3, 4, or more positions). See also Table 3 below.Subcellular localization signals typically require a specificorientation, N or C terminal to the protein to which the signal isattached. A split peroxidase or a fusion protein containing such can befurther fused in frame with a protein tag, which can be any of thoseroutinely used in fusion technology (e.g., Flag and c-Myc) to facilitateprotein expression, detection, and/or purification. A protein tag is apeptide sequence genetically grafted onto the enzyme or the fusionprotein for various purposes, e.g., affinity purification (affinitytag), enhancing solubilization (solubilization tag), or facilitatingchromatography (chromatography tag) or detection (epitope orfluorescence tag). Affinity tags include chitin binding protein (CBP),maltose binding protein (MBP), glutathione-S-transferase (GST), andpoly(His) tag. Solubilization tags include thioredoxin (TRX), poly(NANP)(SEQ ID NO:14), MBP, and GST. Chromatography tags include thoseconsisting of polyanionic amino acids, such as FLAG-tag. Epitope tagsinclude short peptide sequences derived from viral genes, such asV5-tag, c-myc-tag, and HA-tag. Fluorescence tags include GFP and itsvariants.

When necessary, a coding sequence for a split peroxidase can besubjected to codon optimization based on the type of host cells, inwhich the enzyme is to be expressed. For example, when the enzyme is tobe expressed in a mammalian cell, its coding sequence can be subjectedto codon optimization using optimal mammalian codons.

A nucleic acid encoding a split peroxidase or a fusion proteincontaining such can be inserted into a suitable expression vector inoperable linkage to a suitable promoter. An expression vector is oneinto which a desired DNA sequence may be inserted by restriction andligation such that it is operably joined to regulatory sequences and maybe expressed as an RNA transcript. Vectors may further contain one ormore marker sequences (i.e., reporter sequences) suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., beta-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells, hosts,colonies or plaques. Preferred vectors are those capable of autonomousreplication and expression of the structural gene products present inthe DNA segments to which they are operably joined.

As used herein, a marker or coding sequence and regulatory sequences aresaid to be “operably” joined when they are covalently linked in such away as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalprotein, two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequences, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a promoter region would be operablyjoined to a coding sequence if the promoter region were capable ofeffecting transcription of that DNA sequence such that the resultingtranscript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CCAAT sequence, andthe like. Especially, such 5′ non-transcribed regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined coding sequence.Regulatory sequences may also include enhancer sequences or upstreamactivator sequences as desired. The vectors of the invention mayoptionally include 5′ leader or signal sequences. The choice and designof an appropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous nucleic acid, usually DNA, molecules, encoding a splitperoxidase. The heterologous nucleic acid molecules are placed underoperable control of transcriptional elements to permit the expression ofthe heterologous nucleic acid molecules in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pcDNA3.1 (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626-630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303-310, 1996).

In some embodiments, the expression of a split peroxidase or a fusionprotein thereof can be under the control of a cell type/celltissue-specific promoter which drives the expression of a target proteinin a specific type of cells. This is particularly useful, among others,for imaging a particular type of cells in a tissue sample.

Tissue-specific and/or cell type-specific promoters include, but are notlimited to, the albumin promoter (e.g., liver-specific albumin promoter;see Pinkert et al. (1987) Genes Dev 1:268-277); lymphoid-specificpromoters (Calame and Eaton (1988) Adv Immunol 43:235-275), such aspromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748); neuron-specific promoters(e.g., the neurofilament promoter; see Byrne and Ruddle (1989) PNAS86:5473-5477); pancreas-specific promoters (Edlund et al. (1985) Science230:912-916); mammary gland-specific promoters (e.g., milk wheypromoter; see U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166); and developmentally regulated promoters, e.g.,the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev3:537-546).

Either live or fixed cells can be incubated with a peroxidase substratefor a suitable period of time to allow the substrate to be convertedinto a signal-releasing product such as a polymer or a fluorescent dyevia an oxidation reaction catalyzed by the peroxidase when the splitperoxidase reconstitute. Suitable substrates of the split peroxidase,e.g., an APX, are well known in the art. For example, an APX can act onascorbate and other aromatic substrates (e.g., phenol containing,gualacol and salicylhydroxamic acid). In some examples, the peroxidasesubstrates for use in the imaging method described herein isdiaminobenzidine (DAB; including any isoform thereof) or a DAB analog(e.g., 4-chloro-1-naphthol or 3-amino-9-ethylcarbazole; Krieg et al.,2000, Cell Mol. Biol. 46(7):1191-1212; and Baskin et al., 1982, J.Histochemistry & Cytochemistry, 30(7):710-712). In other examples, thesubstrate is a phenol or an aniline.

As used herein, a phenol is a phenyl moiety that is substituted with oneor more —OH, one or more —O⁻, and/or one or more —OH₂ ⁺ groups. Thephenyl moiety may be further substituted with other substituentsincluding, but not limited to, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, —OR^(A1), —N(R^(A1))₂,—SR^(A1), —CN, —C(═NR^(A1))R^(A1), —C(═NR^(A1))OR^(A1),—C(═NR^(A1))SR^(A1), —C(═NR^(A1))N(R^(A1))₂, —C(═S)R^(A1),—C(═S)OR^(A1), —C(═S)SR^(A1), —C(═S)N(R^(A1))₂, —NO₂, —N₃, —N(R^(A1))₃⁺F⁻, —N(R^(A1))₃ ⁺Cl⁻, —N(R^(A1))₃ ⁺Br⁻, —N(R^(A1))₃ ⁺I⁻,—N(OR^(A1))R^(A1), —NR^(A1)C(═O)R^(A1), —NR^(A1)C(═O)OR^(A1),—NR^(A1)C(═O)SR^(A1), —NR^(A1)C(═O)N(R^(A1))₂, —NR^(A1)C(═S)R^(A1),—NR^(A1)C(═S)OR^(A1), —NR^(A1)C(═S)SR^(A1), —NR^(A1)C(═S)N(R^(A1))₂,—NR^(A1)C(═NR^(A1))R^(A1), —NR^(A1)C(═NR^(A1))OR^(A1),—NR^(A1)C(═NR^(A1))SR^(A1), —NR^(A1)C(═NR^(A1))N(R^(A1))₂,—NR^(A1)S(═O)₂R^(A1), —NR^(A1)S(═O)₂OR^(A1), —NR^(A1)S(═O)₂SR^(A1),—NR^(A1)S(═O)₂N(R^(A1))₂, —NR^(A1)S(═O)R^(A1), —NR^(A1)S(═O)OR^(A1),—NR^(A1)S(═O)SR^(A1), —NR^(A1)S(═O)N(R^(A1))₂, —NR^(A1)P(═O),—NR^(A1)P(═O)₂, —NR^(A1)P(═O)(R^(A1))₂, —NR^(A1)P(═O)R^(A1)(OR^(A1)),—NR^(A1)P(═O)(OR^(A1))₂, —OC(═O)R^(A1), —OC(═O)OR, —OC(═O)SR^(A1),—OC(═O)N(R^(A1))₂, —OC(═NR^(A1))R^(A1), —OC(═NR^(A1))OR^(A1),OC(═NR^(A1))N(R^(A1))₂, —OC(═S)R^(A1), —OC(═S)OR^(A1), —OC(═S)SR^(A1),—OC(═S)N(R^(A1))₂, —ON(R^(A1))₂, —OS(═O)R^(A1), —OS(═O)OR^(A1),—OS(═O)SR^(A1), —OS(═O)N(R^(A1))₂, —OS(═O)₂R^(A1), —OS(═O)₂OR^(A1),—OS(═O)₂SR^(A1), —OS(═O)₂N(R^(A1))₂, —OP(═O)(R^(A1))₂,—OP(═O)R^(A1)(OR^(A1)), —OP(═O)(OR^(A1))₂, —S(═O)R^(A1), —S(═O)OR^(A1),—S(═O)N(R^(A1))₂, —S(═O)₂R^(A1), —S(═O)₂OR^(A1), —S(═O)₂N(R^(A1))₂,—SC(═O)R^(A1), —SC(═O)OR^(A1), SC(═O)SR^(A1), —SC(═O)N(R^(A1))₂,—SC(═S)R^(A1), —SC(═S)OR^(A1), —SC(═S)SR^(A1), SC(═S)N(R^(A1))₂,—P(═O)(R^(A1))₂, —P(═O)(OR^(A1))₂, —P(═O)R^(A1)(OR^(A1)), and —P(═O)₂,wherein each occurrence of R^(A1) is independently selected from thegroup consisting of hydrogen, substituted or unsubstituted acyl,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, a nitrogen protecting group when attached to a nitrogenatom, an oxygen protecting group when attached to an oxygen atom, and asulfur protecting group when attached to a sulfur atom, or two R^(A1)groups are joined to form an optionally substituted heterocyclic ring.An example of phenol is hydroxybenzene.

As used herein, an aniline is a phenyl moiety that is substituted withone or more —NH₂, one or more —NH₃ ⁺, and/or one or more —NH⁻ groups.The phenyl moiety may be further substituted with other substituentsincluding, but not limited to, halogen, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, —OR^(B1), —N(R^(B1))₂,—SR^(B1), —CN, —C(═NR^(B1))R^(B1), —C(═NR^(B1))OR^(B1),—C(═NR^(B1))SR^(B1), —C(═NR^(B1))N(R^(B1))₂, —C(═S)R^(B1),—C(═S)OR^(B1), —C(═S)SR^(B1), —C(═S)N(R^(B1))₂, —NO₂, —N₃, —N(R^(B1))₃⁺F⁻, —N(R^(B1))₃ ⁺Cl⁻, —N(R⁻)₃ ⁺Br⁻, —N(R^(B1))₃ ⁺I⁻, —N(OR^(B1))R^(B1),—NR^(B1)C(═O)R^(B1), —NR^(B1)C(═O)OR^(B1), —NR^(B1)C(═O)SR^(B1),—NR^(B1)C(═O)N(R^(B1))₂, —NR^(B1)C(═S)R^(B1), —NR^(B1)C(═S)OR^(B1),—NR^(B1)C(═S)SR^(B1), —NR^(B1)C(═S)N(R^(B1))₂,—NR^(B1)C(═NR^(B1))R^(B1), —NR^(B1)C(═NR^(B1))OR^(B1),—NR^(B1)C(═NR^(B1))SR^(B1), —NR^(B1)C(═NR^(B1))N(R^(B1))₂,—NR^(B1)S(═O)₂RR, —NR^(B1)S(═O)₂OR^(B1), —NR^(B1)S(═O)₂SR^(B1),—NR^(B1)S(═O)₂N(R^(B1))₂, —NR^(B1)S(═O)R^(B1), —NR^(B1)S(═O)OR^(B1),—NR^(B1)S(═O)SR^(B1), —NR^(B1)S(═O)N(R^(B1))₂, —NR^(B1)P(═O),—NR^(B1)P(═O)₂, —NR^(B1)P(═O)(R^(B1))₂, —NR^(B1)P(═O)R^(B1)(OR^(B1)),—NR^(B1)P(═O)(OR^(B1))₂, —OC(═O)R^(B1), —OC(═O)OR^(B1), —OC(═O)SR^(B1),—OC(═O)N(R^(B1))₂, —OC(═NR^(B1)), —OC(═NR^(B1))OR^(B1),—OC(═NR^(B1))N(R^(B1))₂, —OC(═S)R^(B1), —OC(═S)OR^(B1), —OC(═S)SR^(B1),—OC(═S)N(R^(B1))₂, —ON(R^(B1))₂, —OS(═O)R^(B1), —OS(═O)OR^(B1),—OS(═O)SR^(B1), —OS(═O)N(R^(B1))₂, —OS(═O)₂R^(B1), —OS(═O)₂OR^(B1),—OS(═O)₂SR^(B1), —OS(═O)₂N(R^(B1))₂, —OP(═O)(R^(B1))₂,—OP(═O)R^(B1)(OR^(B1)), —OP(═O)(OR^(B1))₂, —S(═O)R^(B1), —S(═O)OR^(B1),—S(═O)N(R^(B1))₂, —S(═O)₂R^(B1), —S(═O)₂OR^(B1), —S(═O)₂N(R^(B1))₂,—SC(═O)R^(B1), —SC(═O)OR^(B1), —SC(═O)SR^(B1), —SC(═O)N(R^(B1))₂,—SC(═S)R^(B1), —SC(═S)OR^(B1), —SC(═S)SR^(B1), —SC(═S)N(R^(B1))₂,—P(═O)(RB)₂, —P(═O)(OR^(B1))₂, —P(═O)R^(B1)(ORB), and —P(═O)₂, whereineach occurrence of R^(B1) is independently selected from the groupconsisting of hydrogen, substituted or unsubstituted acyl, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedcarbocyclyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogenprotecting group when attached to a nitrogen atom, an oxygen protectinggroup when attached to an oxygen atom, and a sulfur protecting groupwhen attached to a sulfur atom, or two R^(B1) groups are joined to forman optionally substituted heterocyclic ring. An example of aniline isaminobenzene.

Examples of the peroxidase substrates for use in the imaging methoddescribed herein include, but are not limited to, those listed in Table2 below:

TABLE 2 Exemplary Peroxidase Substrates Compound classification4-chloro-1-naphthol, Phenol Guaiacol Phenol Pyrogallol Phenol AmplexUltraRed phenol Dihydrofluorescin Phenol p-cresol phenol Dopamine Phenol3-methylphenol Phenol 4-methoxyphenol Phenol 4-hydroxybenzaldehydePhenol 3-amino-9-ethylcarbazole, Aniline DAB Aniline o-phenylenediamine,Aniline 3,3′,5,5′-tetramethylbenzidine, Aniline o-diansidine, AnilineLuminol Aniline 4-aminophthalhydrazide AnilineN-(6-Aminohexyl)-N-ethylisoluminol AnilineN-(4-Aminobutyl)-N-ethylisoluminol Aniline 3-methylaniline Aniline4-methylaniline Aniline 4-methoxyaniline Aniline 5-aminosalicylic acid,Both aniline and phenol 3-methyl-2-benzothiazolinone hydrazone neither2,2′-azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid) (neither)

A substrate is typically provided in an inert, stable, or non-reactiveform, e.g., a form that does not readily react with other molecules inliving cells. Once in contact with an active peroxidase enzyme, thesubstrate is converted from its stable form into a short-lived reactiveform, for e.g., via generation of a reactive moiety, such as a radical,on the substrate by the enzyme. Some substrates are, accordingly, alsoreferred to as radical precursors. The reactive form of the substratethen reacts with and attaches to a molecule, e.g., a protein, in thevicinity of the enzyme. Accordingly, in some embodiments, a substratecomprises an inert or stable moiety that can be converted by the enzymeinto a reactive moiety. The reaction of the substrate with a molecule,e.g., a protein in the vicinity of the enzyme, results in the tagging,or labeling, of the molecule. Typically, a substrate comprises a tag,which is a functional moiety or structure that can be used to detect,identify, or isolate a molecule comprising the tag, e.g., a protein thathas been tagged by reacting with a substrate. Suitable tags include, butare not limited to, for example, a detectable label, a binding agent,such as biotin, or a fluorescent probe, a click chemistry handle, anazide, alkyne, phosphine, trans-cyclooctene, or a tetrazine moiety. Insome embodiments, the reaction of the reactive form of the substratewith a molecule, e.g., a protein, may lead to changes in the molecule,e.g., oxygenation, that can be exploited for detecting and/or isolatingthe changed molecules. Non-limiting examples of such substrates arechromophores, e.g., resorufin, malachite green, KillerRed, Ru(bpy)₃ ²⁺,and miniSOG³¹, which can generate reactive oxygen species that oxidizemolecules in the vicinity of the respective enzyme (reconstituted set ofsplit peroxidases). The oxidation can be used to isolate and/or identifythe oxidized molecules. In some embodiments, the reactive form of thesubstrate crosses cell membranes, while in other embodiments membranesare impermeable to the reactive form of the substrate.

A tag may be, in some embodiments, a detectable label. In someembodiments, a tag may be a functional moiety or structure that can beused to detect, isolate, or identify molecules comprising the tag. A tagmay also be created as a result of a reactive form of a substratereacting with a molecule, e.g., the creation of oxidative damage on aprotein by a reactive oxygen species may be a tag. In some embodiments,the tag is a biotin-based tag and the enzyme—the peroxidase, generates areactive biotin moiety that binds to proteins within the vicinity of theenzyme. In some embodiments, the biotin-based tags are biotin tyramidemolecules. Structures of some exemplary substrates (radical precursors)of peroxidase enzymes that are useful in some of the methods providedherein are provided below:

Additional exemplary peroxidase substrates (radical precursors) areprovided below:

Additional suitable substrates will be apparent to those of skill in theart, and the invention is not limited in this respect. In someembodiments, the tag is an alkyne tyramide and the peroxidase generatesa reactive moiety that binds to proteins within the vicinity of theperoxidase. The alkyne subsequently can be modified, for example, by aclick chemistry reaction to attach a tag (e.g., a biotin tag). The tagcan then be used for further analysis (e.g., isolation andidentification). It should be noted that the invention is not limited toalkyne tyramide, but that any functional group that can bechemoselectively derivatized can be used. Some examples are: azide oralkyne or phosphine, or trans-cyclooctene, or tetrazine, or cyclooctyne,or ketone, or hydrazide, or aldehyde, or hydrazine.

The substrate compounds described herein can be obtained from commercialvendors, e.g., Sigma Aldrich. Alternatively, they can be synthesized bychemistry transformations (including protecting group methodologies),e.g., those described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons(1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and subsequent editions thereof.

Cells expressing a set of split peroxidases, either live or fixed, canbe incubated with a suitable substrate under suitable conditions for asuitable period to allow conversion of the substrate into a product thatreleases a detectable signal, which can then be examined under amicroscope (e.g., an electron microscope or fluorescence microscope) forimaging following routine techniques. See, e.g., Shu et al., PLos Biol.2011, 9(4):e1001041. Utilizing a split peroxidase such as split APEXdescribed herein provides the opportunity not only for EM contact, butalso for colorimetric, fluorescent, and chemiluminescent readouts.

In one example, a split peroxidase (e.g., APEX) fused with a protein ofinterest is expressed in live cells (e.g., mammalian cells). Afterexpression, the cells can be fixed, and then incubated in a solution ofDAB. H₂O₂, can then added into the mixture to allow the peroxidase,which retains activity in fixative, to catalyzes the oxidative reaction,resulting in polymerization of DAB to generate a cross-linkedprecipitate. The cells carrying the DAB polymer thus produced can thenbe incubated with electron-dense OsO4 to generate EM contrast.

In addition to microscopy imaging, any of the split peroxidasesdescribed herein can also be used for various other purposes, includingbioremediation, biocatalysis, diagnostics, biosensors, proteinexpression, transgenics, bioinformatics, protein engineering, andmedical treatment. Processes for performing these uses are well known inthe art. See, e.g., Ryan et al., 2006, Trends in Biotechnology,24(8):355-363.

The invention also includes articles, which refers to any one orcollection of components. In some embodiments the articles are kits. Thearticles include pharmaceutical or diagnostic grade compounds of theinvention in one or more containers. The article may includeinstructions or labels promoting or describing the use of the compoundsof the invention.

As used herein, “promoted” includes all methods of doing businessincluding methods of education, hospital and other clinical instruction,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with compositions ofthe invention.

“Instructions” can define a component of promotion, and typicallyinvolve written instructions on or associated with packaging ofcompositions of the invention. Instructions also can include any oral orelectronic instructions provided in any manner.

Thus the agents described herein may, in some embodiments, be assembledinto research, pharmaceutical or diagnostic kits to facilitate their usein research, diagnostic or therapeutic applications. A kit may includeone or more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendeduse of these agents for labeling in in vitro or in vivo or in othersamples such as cell lysates.

The kit may be designed to facilitate use of the methods describedherein and can take many forms. Each of the compositions of the kit,where applicable, may be provided in liquid form (e.g., in solution), orin solid form, (e.g., a dry powder). In certain cases, some of thecompositions may be constitutable or otherwise processable (e.g., to anactive form), for example, by the addition of a suitable solvent orother species (for example, water or a cell culture medium), which mayor may not be provided with the kit. As used herein, “instructions” candefine a component of instruction and/or promotion, and typicallyinvolve written instructions on or associated with packaging of theinvention. Instructions also can include any oral or electronicinstructions provided in any manner such that a user will clearlyrecognize that the instructions are to be associated with the kit, forexample, audiovisual (e.g., videotape, DVD, etc.), Internet, and/orweb-based communications, etc.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a cell or a subject. Thekit may include a container housing agents described herein. The agentsmay be prepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are sued, the liquidform may be concentrated or ready to use.

The kits, in one set of embodiments, may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the containers may comprise a positive control for anassay. Additionally, the kit may include containers for othercomponents, for example, buffers useful in the assay.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1: Split HRP Fragments are Active Once Associated

Experiments were performed to identify split HRP fragment pairs thatreconstitute in a manner that is dependent on a protein-proteininteraction (FIG. 1). The two fragments of HRP were separately fused tothe C-termini of FRB and FKBP, respectively, with flexible, 12-aminoacid linkers (consisting of glycine, serine, and threonine residues)separating FRB or FKBP from the fragments of HRP. The fusion constructswere generated using standard molecular biology techniques. Once theconstructs were generated, they were transfected together into culturedmammalian cells. The cells were then cultured in the presence or absenceof the drug rapamycin, which induces a tight protein-protein interactionbetween FRB and FKBP. Cells were labeled with a fluorogenic peroxidasesubstrate to determine which conditions give rise to reconstitution ofperoxidase activity.

The data is shown in FIG. 1. These constructs possess N-terminalsecretion signal sequences and C-terminal KDEL (SEQ ID NO:7) sequences,which leads to expression in the lumen of the endoplasmic reticulum.This ER localization is important because HRP fails to become activeoutside the secretory pathway. The approach is useful for determiningwhich fragment pairs function best with a protein-protein interactionfor assembly into an active form.

Similar data on split APEX for detection of protein-protein interactionsis presented in FIG. 6. Use of split APEX instead of split HRP, providesan additional advantage, that the fragments do not need to be confinedto the secretory pathway.

Example 2: Split HRP is More Sensitive than Split GFP

Live HEK293T cells (a type of cultured mammalian cell) were transfectedwith complementary split HRP fragment pairs (FIG. 2). Pair 3 correspondsto the cut site after amino acid 58, and pair 11 corresponds to the cutsite after amino acid 213. The sHRP fragments were fused to FRB andFKBP, as shown in FIG. 8. As a control, full-length HRP was transfectedinto a different set of cells. Cells were cultured overnight aftertransfection at 37 degrees C. in the presence or absence of the drugrapamycin. Live cells were treated with a solution containing Amplex Red(50 uM) and hydrogen peroxide (6.67 mM) in DPBS (a buffer that isrelated to phosphate buffered saline, but it contains some extranutrients). After 10 minutes, the amplex solution was removed from thecells and replaced with DPBS. Cells were then imaged using a confocalmicroscope while alive to detect resorufin fluorescence, which indicatesperoxidase activity. The data is shown in FIG. 2. Split HRP pairsproduce a robust signal.

Example 3: Inter-Cellular Reconstitution of HRP is Dependent on theProtein-Protein Interaction Between Neuroligin (NLG) and Neurexin(NRX3B)

HEK293T cells were transfected with fragment pair 3, fragment pair 11,or full-length HRP as in Example 2. In this experiment, cells werecultured at 30 degrees C. in the presence or absence of rapamycin (FIG.3). Cells were fixed chemically using formaldehyde, washed, then treatedwith diaminobenzidine (DAB, 0.5 mg/mL) with 10 mM H2O2 in cold PBSbuffer for 20 minutes. Cells were then washed and imaged by bright fieldto detect the DAB polymer. This DAB polymer is useful for electronmicroscopy, since it becomes electron dense after treatment with osmiumtetroxide. The data is shown in FIG. 3 and demonstrates that split HRPcan be useful for electron microscopy.

Example 4: SplitHRP for Ultra-Sensitive Synapse Detection

FIG. 4 demonstrates the overall scheme of how split HRP can be appliedfor synapse detection. One fragment of HRP is targeted to thepresynapse, and the other fragment is targeted to the post-synapse. Thefragments bind to each other and form into an active, reconstituted formas synapses only. Upon treatment with a peroxidase substrate, label isdeposited specifically at synapses. A variety of peroxidase substratescan be employed in this scheme. Since HRP is an enzyme that gives largeamplification of signal, even if only a few copies of HRP arereconstituted at a specific synapse, the signal should still bedetectable. This signal amplification gives split HRP a distinctadvantage over split green fluorescent protein, which has been appliedfor synapse detection as well, but has given fluorescence that is toodim for many applications.

Example 5: SplitHRP for Labeling Synaptic Cleft

FIG. 5 demonstrates how split HRP works for synapse detection incultured neurons. Cultured rat hippocampal neurons were transientlytransfected with HRP fragment 1 along with a blue fluorescent proteinmarker at DIV10. At DIV11, the same set of cells was transientlytransfected with HRP fragment 2 along with a green fluorescent proteinmarker. Because of the low transfection efficiency in each step and therandomness of transient transfection, distinct sets of cells aretransfected in each step. At DIV12, cells were fixed chemically usingformaldehyde, then washed and treated with biotin-phenol (the substrateused in Rhee et. al. 2013, Science, for proteomics). After washing andtreatment with avidin conjugated to a fluorophore, cells were imaged byconfocal microscopy. The data is shown in FIG. 5. Fluorescent avidinlabeling was detected only at contact sites between green and blueneurons, indicating that split HRP can be effectively employed forsynapse detection.

Example 6: Rapamycin Dependence of Activity for 2 Promising Split APEXPairs

FIG. 7 shows how HEK293T cells were transfected with complementary splitAPEX fragments fused to FRB or FKBP, as described in earlier figures forsplit HRP. Cells were cultured in the presence or absence of rapamycin,then labeled using Amplex Red and imaged while alive, as explained inearlier figures for split HRP. The data is shown in FIG. 7. The splitAPEX “2” cut site in the figure corresponds to cutting after amino acidposition 50, and the cut site “12” corresponds to cutting after aminoacid position 200. In this experiment, constructs were generated inwhich the split APEX fragments are targeted to either the mitochondrialmatrix or the cytosol. Split APEX can become active in both of thesecompartments, demonstrating its applicability in compartments whereother split labels do not work.

Example 7: Both Split HRP Fragments are Required for Activity

In this experiment, HEK293T cells were transiently transfected withconstructs encoding split HRP amino acids 1-213:

split HRP amino acids 214-308: SEQ ID NO: 13 QLTPTFYDNSCPNVSNIVRDTIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRTEKDAFGNANSARGFPVIDRMKAAVESACPRTVSCADLLTIAAQQSVTLAGGPSWRVPLGRRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKNQCRFIMDRLYNFSNTGLPDPT LNTTYLQTLRGLCPLNG,(SEQ ID NO: 15) NLSALVDFDLRTPTIFDNKYYVNLEEQKGLIQSDQELFSSPNATDTIPLVRSFANSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRVVNSNS,or both (FIG. 8). The split HRP fragments in this experiment werelocalized to the ER lumen using the approach described in FIG. 1, and inthis case, the fragments were not attached to FRB and FKBP—the fragmentswere simply free-floating in the ER lumen. Cells were labeled whilealive with amplex red, then fixed using formaldehyde, then washed andimmunostained to detect expression of the split HRP fragments. Theresorufin fluorescence was detectable after fixation, as observedpreviously in Martell et. al. 2012 Nature Biotechnology. The data isshown in FIG. 8. These data indicate that the split HRP 213 fragmentsreconstitute spontaneously in the ER lumen of HEK293T cells, without anyassistance from a protein-protein interaction. Both fragments arerequired for activity.

Example 8: Split HRP is More Sensitive than Split GFP

In this experiment, cultured neurons were transfected and labeled asdescribed for FIG. 5. Split HRP gives strong and easily detectablefluorescent signal at contact sites between the two transfected pools ofneurons. In a side-by-side comparison, constructs encoding split GFPwere generated and introduced to cultured neurons using the sameprocedure. The two different pools of neurons were marked using a redfluorescent protein marker or far red-colored antibody staining. Thedata is shown in FIG. 9. GFP signal, was not detectable at contact sitesbetween the two pools of neurons at a matched intensity scale to thesplit HRP images. When a much higher contrast level was used, split GFPlabeled was indeed detectable at the contact sites, although it was notmuch brighter than background fluorescence. These data indicate thatsplit HRP is much more sensitive than split GFP for fluorescent labelingof synapses in cultured neurons.

Example 9: Inter-Cellular Reconstitution of HRP is Dependent on theProtein-Protein Interaction Between Neuroligin (NLG) and Neurexin(NRX3B)

FIG. 10 give more detail on the constructs used in FIG. 9. Thepre-synaptic construct used for split HRP synapse detection has thesHRPa fragment (amino acids 1-213: SEQ ID NO:13) fused to the N-terminusof neurexin3 beta (NRX3B), and the post-synaptic construct has the sHRPb(amino acids 214-308: SEQ ID NO:15) fused to the N-terminus ofneuroligin (splice variant). The data is shown in FIG. 10. Neurexin andneurologin are known to interact with low nanomolar binding affinity inan intercellular fashion across the synaptic cleft. This tightprotein-protein interaction is required for split HRP reconstitutionintercellularly, because when the two split HRP fragments are bothattached to neurexin (a negative control, because neurexin does not bindto itself intercellularly), no peroxidase activity is observed. Thisdependence on a protein-protein interaction is advantageous for thesplit HRP system, since it is desirable to have reconstitution only atsynapses, and not at random incidental contact sites between the splitHRP fragments that are not driven by a synaptic protein-proteininteraction.

Example 10: Split HRP Activity Survives Chemical Fixation and a Varietyof Permeabilization and Tissue Blocking Treatments

FIG. 11 shows cultured neurons that were transfected in “Cis” with thesplit HRP fragments along with a green fluorescent protein marker. Bothsplit HRP fragments and the FP plasmid were transfected on the same day,causing a small subset of neurons to become transfected. All transfectedneurons carry all 3 plasmids. This causes the split HRP fragments tofind each other all across the plasma membrane of the transfectedneurons, so when the peroxidase labeling is performed (usingbiotin-phenol after fixation, as described for FIG. 5), fluorescence isobserved everywhere on the surface of the transfected neuron, withoutspecificity for synapses.

The data is shown in FIG. 11. The experiment was conducted to testwhether split HRP activity could survive a variety of permeabilizationand blocking conditions that are useful for tissue labeling. Forexample, treatment with high concentrations of H₂O₂ inactivateendogenous peroxidases, thus decreasing background labeling. Treatmentwith 33 mM H2O2 inactivates endogenous peroxidases in tissue to decreasebackground labeling (this is called “blocking” of tissue).Permeabilization with triton or methanol improves accessibility ofperoxidase small molecule substrates into the tissue interior, thusimproving the labeling efficiency. Triton and methanol are commonly-usedchemical treatments for permeablization of cells to improve access ofsmall-molecule substrates to the cell interior. Fortunately, split HRPactivity survives all of these treatments. The experiment demonstratesthe ability of split HRP activity to survive these treatments, making itparticularly attractive for applications in tissue.

Example 11: Split APEX Staining with DAB, which Gives Contrast forElectron Microscopy

Cells were transfected with: HaloTag-FRB-sAPXa; HaloTag-FKBP-sAPXb andNuclear YFP. Similar to FIG. 3, but using split APEX instead of splitHRP, the technology was demonstrated to be useful in electron microscopybased assays (FIG. 12). The transfected, rapamycin incubation, and Dablabeling were performed in the same way. The images shown in FIG. 12demonstrated that split APEX activity is able to survive chemicalfixation and is detectable using DAB, a substrate that gives contrastfor electron microscopy. Therefore, split APEX can be used with electronmicroscopy.

Example 12: Split “APEX2”, which is Derived from an Improved Version ofAPEX, Performs Better than the Original Split APEX

HEK293T cells were transiently transfected, then cultured overnight inthe presence or absence of rapamycin or the heme cofactor. Cells werecultured at 37 degrees C. The next day, cells were labeled while aliveusing Amplex Red, then the amplex solution was removed, and live cellswere imaged using confocal microscopy.

Certain “cut sites” on APEX give rise to fragment pairs that canreconstitute to give peroxidase activity. This data is shown in FIG. 13.An improved version of APEX has been engineered, which is called“APEX2.” This enzyme has only 1 mutation relative to APEX, but greatlyimproves the brightness of labeling for all applications tested so far.

(SEQ ID NO: 12) GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELG FADA 

The data demonstrates that split APEX2 constructs performed much betterthan split APEX.

Example 13: Split APEX2 Using Biotin-Phenol as the Labeling Substrate:Potential for Proteomic Labeling Applications

Cells were transfected and treated with or without rapamycin asdescribed for FIG. 13. In this case, live cells were incubated withbiotin phenol (following the procedure used for proteomics labeling, asreported by Rhee et. al. 2013, Science), then treated for 1 minute with1 mM H2O2, then fixed, permeablized, and stained with afluorophore-conjugated avidin. FIG. 14 shows that split APEX2 givesrobust biotin phenol labeling in the presence of rapamycin, but not inthe absence. The data demonstrates the utility of split APEX2 inproteomics applications.

Example 14: Characterizing the Kinetics of Rapamycin Response for SplitAPEX2

HEK293T cells were transiently transfected with complementary splitAPEX2 fragments (corresponding to the cut site after amino acid 89).Cells were cultured at 37 degrees C. overnight in the presence ofrapamycin for varying lengths of time, and with or without 2 uM heme forthe entire night. The next day, living cells were washed briefly inbuffer, then treated with Amplex Red containing 6.67 mM H2O2. After 15minutes, fluorescence from resorufin (the product of a peroxidase/H2O2reaction with Amplex Red) was detected in the cell supernatant.

The data is shown in FIG. 15. The detected fluorescence gives anindication of intracellular peroxidase activity; although APEX2reconstitution occurred inside the cell in the cytoplasm, thefluorescent product resorufin leaks into the extracellular media and ishence detectable using a plate reader. These data indicate that splitAPEX2 gives a detectable change in peroxidase activity at least after 20minutes of rapamycin treatment.

Example 15: Screening for Best Cut Site in HRP

In order to confirm that multiple split pairs were useful according tothe methods of the invention 19 different cut sites were generated andtested. The pairs were co-expressed in the ER lumen and cells werelabeled using Amplex Red. Fluorescence microscopy was used to detectactivity.

FIG. 16A depicts a set of bars representing the amino acid sequence ofhorseradish peroxidase, or HRP (308 amino acids long). The cysteineresidues are pointed out, and lines connecting the cysteine residuesrepresent intramolecular disulfide bonds within HRP. The arrows pointingto the HRP sequence represent the approximate locations of cut sitestested. In order to test 1 specific cut site, two constructs of thefollowing form were generated:

Secretion signal-FRB-split HRP fragment (N-terminal)-KDEL (SEQ ID NO:7)

Secretion signal-FKBP-split HRP fragment (C-terminal)-KDEL (SEQ ID NO:7)

The secretion signal and the C-terminal KDEL (SEQ ID NO:7) sequenceserved to localize the constructs to the lumen of the endoplasmicreticulum. To test a specific cut site, two complementary constructswere transfected into HEK293T cells and cultured in the presence orabsence of the drug rapamycin overnight. Rapamycin induces a tightprotein-protein interaction between FRB and FKBP. Screening in both thepresence and absence of rapamaycin determine which complementaryfragments spontaneously assemble into an active form and which fragmentpairs require a protein-protein interaction to bring them together anddrive the reconstitution.

The images shown in FIG. 16B are from cells labeled while alive withAmplex UltraRed (50 uM) with hydrogen peroxidase (6.67 mM) in colorlessbuffer, then imaged while alive. The fluorescent product, resorufin, isa bright red fluorophore that fills the entire cell and also leaks intothe extracellular buffer to some extent. Whether reconstitutedperoxidase activity was present within the cells can be determined usingconfocal imaging for resorufin, or by quantifying resorufin fluorescence(excitation 568, emission 581) using a plate reader—which allows formore convenient screening of many constructs and conditions.

FIG. 16C depicts a three dimensional crystal structure for full-lengthHRP with cut site 213. The amino acid sequence of the split HRP fragmentpair that provided the most robust reconstitution, of those tested thusfar, i.e., the brightest fluorescence when the cells were cultured inthe presence of rapamycin are created by cut site 213 (sHRPa havingamino acids 1-213 and sHRPb having amino acids 214-308). Note that forthis fragment pair to reconstitute into an active form, a disulfide bondneeds to be formed intermolecularly between the two fragments.

Example 16: Temperature and Rapamycin Dependence of Activity for 7 Pairs

In this example, 7 fragment pairs that showed promise in the initialscreen were examined more closely. The data is shown in FIG. 17. In thiscase, cells were cultured in the presence or absence of rapamycin, andat either 30 degrees C. or 37 degrees C. The cell labeling procedure wasthe same as described in Example 15 (50 uM Amplex Ultra Red, 0.02% H2O2,25 min labeling on live HEK cells). Some split protein systems, such assplit YFP reported by Kerpolla and co-workers (called “BifC”), fail togive any signal if cells are cultured at 37 degrees C., and they onlybecome functional at 30 degrees C.

The data is shown in FIG. 17. The majority of the fragment pairs gave nodetectable fluorescence when the cells were cultured at 37 degrees C.,although several had the desirable property of being rapamycin-dependentfor reconstitution at 30 degrees C. Two fragment pairs gave brightfluorescence at 37 degrees C., which is the more physiologicallyrelevant condition for mammalian cells (and hence these are the twofragment pairs focused on in further studies). In this dataset, fragmentpair 58 appears to be rapamycin-dependent for reconstitution, whilefragment pair 213 is not dependent on rapamycin. Each of these fragmentpairs has utility, depending on the type of assay being conducted. Forinstance, split pair 58 is particularly useful for detection ofprotein-protein interactions, while split pair 213 is not as useful forthis purpose, because the fragments spontaneously assemble regardless ofwhether a protein-protein interaction is bringing them together. For anapplication such as split HRP for synapse detection the fragment pair213 is actually preferable. In the context of synapse detection, thesplit HRP 213 fragments do not spontaneously assemble; a protein-proteininteraction is required to drive their assembly. The likely reason forthis difference in requirement for a protein-protein interaction is thatthe concentrations of the two split HRP fragments are much lower whenthey meet intercellularly across the synaptic cleft, as opposed to whenthey meet inside the ER lumen with both fragments overexpressed withinthe same cell. Different cell types from different organisms areoptimally cultured at different temperatures. For cells cultured at 30degrees C. or lower, while many fragment pairs will be useful, fragmentpairs other than 58 or 213 may be even more preferred.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. An isolated component of a split peroxidase,wherein the split peroxidase is two fragments, a first fragment and asecond fragment, which together form a whole peroxidase with a cleavagesite, comprising the first fragment of the whole peroxidase fused to aprotein of interest, optionally through a linker, wherein the cleavagesite is a solvent exposed loop region of the whole peroxidase.
 2. Theisolated component of a split peroxidase of claim 1, wherein the proteinof interest is a mitochondrial protein, mitochondrial matrix protein, amitochondrial intermembrane space protein, a mitochondrial innermembrane protein, a mitochondrial outer membrane protein (facingcytosol), a Golgi protein, an endoplasmic reticulum lumen protein, anendoplasmic reticulum membrane protein (facing cytosol), a cell surfaceprotein, a secreted protein, a nuclear protein, a vesicle protein, acell skeleton protein, a cell skeleton-binding protein, a motor protein,a gap junction protein, a chromatin-organizing protein, a transcriptionfactor protein, a DNA polymerase protein, a ribosomal protein, asynaptic protein, or an adhesion protein.
 3. The isolated component of asplit peroxidase of claim 1, further comprising a cellular localizationsignal peptide linked to the split peroxidase or the protein ofinterest.
 4. The isolated component of a split peroxidase of claim 3,wherein the cellular localization signal peptide is an ER-targetingsignal peptide, a Golgi-targeting signal peptide, amitochondria-targeting signal peptide, a nuclear localization signalpeptide, or a nuclear export signal peptide and/or wherein the cellularlocalization signal peptide comprises an amino acid sequence selectedfrom the group consisting of: (SEQ ID NO: 4) DPVVVLGLCLSCLLLLSLWKQSYGGG,(SEQ ID NO: 5) MLATRVFSLVGKRAISTSVCVRAH, (SEQ ID NO: 6) LQLPPLERLTLD,and (SEQ ID NO: 7) KDEL.


5. The isolated component of a split peroxidase of claim 3, wherein theperoxidase has an amino acid sequence selected from SEQ ID NO:1, 2, 3,8, 9, 10, 11, or 12 and wherein the linker is a flexible amino acidlinker.
 6. An isolated component of a split peroxidase, wherein thesplit peroxidase is two fragments, a first fragment and a secondfragment, which together form a whole peroxidase with a cleavage site,comprising the first fragment of the whole peroxidase fused to acellular localization signal, wherein the cleavage site is a solventexposed loop region of the whole peroxidase.
 7. The isolated componentof a split peroxidase of claim 6, wherein the cellular localizationsignal peptide is an ER-targeting signal peptide, a Golgi-targetingsignal peptide, a mitochondria-targeting signal peptide, a nuclearlocalization signal peptide, or a nuclear export signal peptide.
 8. Theisolated component of a split peroxidase of claim 6, wherein thecellular localization signal peptide comprises an amino acid sequenceselected from the group consisting of: (SEQ ID NO: 4)DPVVVLGLCLSCLLLLSLWKQSYGGG, (SEQ ID NO: 5) MLATRVFSLVGKRAISTSVCVRAH,(SEQ ID NO: 6) LQLPPLERLTLD,  and (SEQ ID NO: 7) KDEL.


9. The isolated component of a split peroxidase of claim 1, wherein theperoxidase has an amino acid sequence fragment selected from SEQ IDNO:1, 2, 3, 8, 9, 10, 11, or
 12. 10. The isolated component of a splitperoxidase of claim 1, wherein the split peroxidase is SEQ ID NO: 13 or15.
 11. The isolated component of a split peroxidase of claim 1, whereinthe split peroxidase is selected from the group consisting of aminoacids 1-58 of SEQ ID NO: 8, amino acids 1-308 of SEQ ID NO: 8, aminoacids 1-213 of SEQ ID NO: 8, amino acids 214-308 of SEQ ID NO: 8, aminoacids 1-50 of SEQ ID NO:11, amino acids 51-249 of SEQ ID NO:11, aminoacids 1-200 of SEQ ID NO:11, amino acids 201-249 of SEQ ID NO:11, aminoacids 1-50 of SEQ ID NO:12, amino acids 51-249 of SEQ ID NO:12, aminoacids 1-200 of SEQ ID NO:12, or amino acids 201-249 of SEQ ID NO:12. 12.A kit, comprising: a set of split peroxidase components, wherein thesplit peroxidase components are two fragments, a first fragment and asecond fragment, which together form a whole peroxidase with a cleavagesite and wherein the cleavage site is a solvent exposed loop region ofthe whole peroxidase, and instructions for delivering the splitperoxidase components to a cell to label one or more proteins of thecell.
 13. The kit of claim 12, further comprising a substrate.